Immunological and biological applications of anti-progesterone antibodies

Abstract

Seven hybridoma cell lines, 051-01 (common name : 15A, IgG$_1$), 051-02 (common name : 28A, IgG$_{2b}$), 051-03 (IgG$_1$), 051-04(IgM), 051-05 (IgG$_{2a}$), 051-06(IgM), and 051-07(IgG$_{2a}$) producing monoclonal antibodies to progesteron were established. Progesterone 11$\alpha$ -hermisuccinate-bovine serum albumin conjugate, prepared using carbodiimide, was used to immunize BALB/c mice. The spleen cells from the immunized mice were taken to fuse with mouse myeloma (P3-X63-Ag8.654) cells. The hybridoma cells thus obtained were screened by radioimmunoassay using [$^3$H]-progesterone. The clones 051-01, 051-03, and 051-05 were purified using protein A-Sepharose 4B affinity chromatography utilizing their subtype characteristics. The purity of the isolated IgG was identified by SDS-PAGE and capillary electophoresis. The affinity constant ranged from 5 $\times$ 10$^7$M$^{-1}$ to 4 $\times$ 10$^8$M$^{-1}$ and the progesterone binding capacity of the monoclonal antibodies from 7 to 72 pmole/mg IgG by the Scatchard analysis method. The cross-reactivity of the monoclonal antibodies, 15A and 28A with other steroid hormones has shown that these antibody reactions were highly specific and sensitive in a competitive immunoassay. To develope EIA system, the influence of the conjugation site on the specificity of monoclonal antibodies to progesterone and on the performance of enzyme immunoassay was assessed. Monoclonal antibodies against progesterone conjugated to carrier protein through substituent on the A-ring (C-3 position) or the C-ring (C-11 position) of progesterone were used in the enzyme immunoassay. Antibody specificities were determined by testing the ability of 11 representative steroids to displace labelled progesterone in a competitive enzyme immunoassay and a radioimmunoassay (RIA). Immunization with progesterone conjugated to BSA through substituent on the A-ring (C-3) resulted in the formation of monoclonal antibody (mAb) which fairly specific for progester...